Probiotic Bacteria on

Aquaculture
 Lecture Outline
 Introduction/Definition of Probiotics
 Fundamental Questions
 Recent Findings
 Possible Modes of Action
 Rationale for Selecting Probiotics
 Conclusions and Further Directions

Source of notes: Verschuere, L., Rombaut, G., Sorgeloos, P., and

Verstraete, W., 2000. Probiotic bacteria as biological control agents in
aquaculture. Microb. Mol. Biol. Rev., 64(4):655-671.
 Introduction
 Aquaculture is growing faster than beef cattle
production (10% vs. 3%)
 Epizootics (disease) = major limiting factor in
fish/shrimp developments!
 Disinfectants, antimicrobial drugs have had little effect
in controlling disease.
 Massive use of antimicrobials increases selective
pressure on microbes and encourages natural
emergence of bacterial resistance
 Resistant bacteria thrive after non-resistant strains have
been killed and can even pass on resistance genes to
other bacteria that have not been exposed to antibiotics
 Introduction
 Emphasis should be place on prevention
 More cost-effective than cure!
 Antimicrobials, disinfectants and pesticides largely
treat symptoms of the problem and not the cause
 Alternative strategies are just catching on
 Example: via vaccination, Norway has reduced
chemical therapeutant use from 50 MT in 1987 to
less than 747 kg in 1997 (with 7x concomitant
production increase!)
 Other: use of immunostimulants w/or w/out
vaccines
HOW DO DISEASE EMERGE
 Introduction
 Use of bacteria as a food source and as a biological
control agent of fish disease was first proposed by
Yasuda and Taga (1980)
 Vibrio alginolyticus has been used as a probiotic in
shrimp hatcheries in Ecuador since 1992
 Reduced hatchery down time from 7 days per month
to less than 21 days per year!
 FAO has now designated use of probiotics as a major
means for improvement of aquatic environmental
quality
 Ultimate goal: make aquaculture products more
acceptable to consumers
Definition of Probiotics
 Many proposed, however: a live microbial feed
supplement which beneficially affects the host animal
by improving its intestinal balance
 Historically: terrestrial animals, genus Lactobacillus
 Definition (above) requires some additional
considerations:
– 1) bacteria in aquatic medium influence composition of gut
microbiota and vice versa
– 2) immediate ambient environment has much greater
influence on microbiota than with terrestrials
 In aquatic environments, hosts and microorganisms
share the ecosystem
 Terrestrials: the gut represents a moist habitat in a
water-limited world
Definition of Probiotics
 Big Issue 1: aquatics are surrounded by an environment
supporting their pathogens independently of the host animal
 Result: opportunistic pathogens can reach high densities
around the fish/shrimp
 Surrounding bacteria are commonly ingested with the feed or
via drinking (maximum case: filter feeders)
 Research in probiotics began with fish juveniles but more
attention recently given to shrimp and finfish larvae
 Big Issue 2: terrestrials have inherent colonizing bacteria from
the mother, aquatics largely spawned as axenic eggs (no further
contact with parent)
 Ambient bacteria colonize eggs surface, young larvae often have
no developed gut (e.g., shrimp), no microbial community in gut,
gills or skin
 Point: properties of bacteria in ambient water are very
important
 Definition of Probiotics
 Better definition: a live microbial adjunct which has a
beneficial effect on the host by modifying the host-associated
or ambient microbial community, by insuring improved use of
feed or by enhancing its nutrition, by enhancing the host
response towards disease, or by improving quality of the
ambient environment
 Our focus: response towards disease and improvement of the
ambient environment
 Jobs of Microbial Adjuncts:
– 1) microbial adjuncts preventing proliferation of pathogens in gut or
elsewhere;
– 2) improved digestibility;
– 3) deliver improved nutrition to aquatics;
– 4) enhancing host response to disease (acquired);
– 5) improved environmental quality.
 Can you Manipulate
Microbial Communities?
 Difficult: 1) discontinuous culture cycles; 2)
disinfection during pond prep; 3) sudden increases in
nutrients due to feeding
 Unlikely under intensive rearing
 Must consider deterministic factors (known
response): salinity, temp, quality/quantity of feed
 Point: the environment selects the range of microbes
(axiom of environmental selection)
 Stochastic factors: chance, right place/right time
 Evidence: identical cultures started simultaneously
yield different assemblages
 Can you Manipulate
Microbial Communities?
 Concept: instead of accidental colonization, start with a
probiotic that is well adapted to prevailing environmental
conditions
 This is probably better than competing with a dominant, well-
established, non-probiotic
 Long-term exposure is often required to achieve a probiotic
effect
 Does the probiotic have to be continuously introduced to the
culture?
 Evidence: in most cases, yes (at least with Lactobacillus sp.)
 Most fish contain a specific intestinal microbiota established at
the juvenile stage
 Unless the host has been exposed to a limited range of
microorganisms in its development, a single addition won’t
result in long-term colonization
 Recent Findings
 As mentioned, it all started with Yasuda and
Taga (1980);
 Usually added in feed or to culture water as
preventative agents against infection by
pathogenic bacteria
 Nutritional benefits are usually secondary
 Typical genera: Lactobacillus, Vibrio, Bacillus,
Pseudomonas
 The following is a summary of findings based
on various aquatic species
 Recent Findings: fish
eggs/larvae
 For incubators, use of antibiotics must be minimal
 Antibiotics don’t represent control; instead, unfavorable
alteration of microbiota
 Goal: establish colonization on the egg prior to pathogen
colonization (i.e., no substrate)
 This, in turn, affects subsequent gut colonization
 Once initial feeding has started, probiotics typically added
to culture water or culture medium of live feed items
(e.g., algae, rotifers, etc.)
 Result: improved survival, faster growth
 Mechanism? Production of antibiotics or siderophores
(metal sequesterers)
Recent Findings: finfish
 Digestive tract of finfish contains 108 cells/g (Ringo et al.,
1995)
 For cod, Gadus gadus, gut is colonized by similar bacteria
as found in eggs (Hansen and Olafsen, 1999)
 Putative probiotics added as soon as possible after
hatching in order to colonize gut prior to feeding (Ringo
and Vadstein, 1998)
 Turbot and dab harbor bacteria capable of suppressing
growth of V. anguillarum (Ollson et al., 1992)
 V. alginolyticus was effective in reducing disease caused
by Aeromonas salmonicida in Atlantic salmon (Austin et
al., 1995)
 Recent Findings: finfish
 Kennedy et al. (1998) showed addition of a Gram-positive
probiotic increased survival, size uniformity, and growth rate of
snook, red drum, spotted sea trout and striped mullet.
 Gram et al. (1999) reported a strain of Pseudomonas
fluorescens reduced mortality of 40 g rainbow trout infected
with pathogenic V. anguillarum
 Garcia-de-la-Banda et al. (1992) added Streptococcus lactis and
Lactobacillus bulgaricus to rotifers and Artemia sp. nauplii and
recorded 6x higher survival at weaning than untreated controls
 Nikoskelainen et al. (2003) showed immune enhancement in
rainbow trout via Lactobacillus rhamnosus supplemented in
feeds
 Recent Findings: shrimp
 Broad application in hatcheries, but few in-depth studies
published
 often used as food source (e.g., soil bacteria for P. monodon
nauplii)
 improved survival (57% vs. 0%) after 13 days against V.
anguillarum
 improved survival of L. vannamei PL’s inoculated with V.
alginolyticus (non-pathogenic) vs. oxytet and control (Garriques
and Arivalo, 1995)
 Griffith (1995) reported that following the introduction of
probiotics in Ecuador in 1992, hatchery down-time between
batches was reduced from 7 days per month to 21 days per year,
production volumes increased by 35% and antimicrobial use
decreased by 94%
 In shrimp hatcheries in New Caledonia, a strain of
Pseudoalteromonas piscicida was found to inhibit the growth of
Vibrio sp. (Saulnier et al., 2000)
Recent Findings: bivalave
molluscs
 Most research has focused on nutritional
contributions to mollusc larvae
 most work in vitro wherein autochthonous
strains have been isolated from scallops
and have shown some inhibition to Vibrio
sp. and Aeromonas hydrophila
 Bacillus sp. and Lactobacillus sp. shown to
depurate oysters (Crassostrea virginica)
against V. vulnificus (Williams et al., 2001)
Part II. Modes of Action of
Probiotic Bacteria
 Modes of Action
 Many publications about probiotics have
emerged in aquaculture in the last decade
 most based on empirical arguments (simple)
 modes of action were largely circumstantial
 several modes of action have been proposed
as a result of human and agricultural
applications
 Modes of Action
 Info on terrestrial investigations has been used for
aquatics (esp. Lactobacillus sp.)

 one problem: distinction between 1) the intrinsic ability of

the probing to positively influence the host and 2) its
ability to reach and maintain itself in the location where
the effect is to be exerted

 Point: does not matter if it produces siderophores or

inhibitory compounds in the gut if it’s never ingested

 can be very important: Mytilus edulis (Blue Muscle) can

selectively ingest/digest microbes
 Modes of Action
 Also, if the candidate probiont cannot
proliferate in the gut it probably won’t
exert a strong effect
 will need continuous application
through the diet or via the water ($$$)
 summary: they must reach the
location where effect is desired
 Possible Modes of Action
 production of inhibitory compounds
 competition for chemicals/available
energy
 competition for adhesion sites (exclusion)
 enhancement of the immune response
 improvement of water quality
 interaction with phytoplankton
 a source of macro- and micro-nutrients
 enzymatic contribution to digestion
 (1) production of
inhibitory compounds
 Release of chemicals having a bactericidal or
bacteriostatic effect
 ultimate result: competitive edge for
nutrients/energy
 production sites: in host intestine, on its
surface, or in culture medium
 products: antibiotics, bacteriocins,
siderophores, lysozymes, proteases, hydrogen
peroxide, organic acids (pH change)
 exact compound is seldom identified: hence, the
term “inhibitory”
 (1) production of
inhibitory compounds
 Lactobacillus sp. produces bacteriocins (toxins)
 marine bacteria produce bacteriolytic enzymes
against V. parahaemolyticus
 Alteromonas sp. produces monastatin, shown
to be inhibitory against Aeromonas hydrophila
 inhibitory effects have been shown by
probiotics against aquaculture pathogens
 no demonstration under in vivo conditions
(oops!)
 more research required!!! (Didn’t you mention this last time??)
(2) Competition for Chemicals
or Available Energy
 Explains how different microbial populations exist in
same ecosystem
 it is likely that it occurs in the mammalian gut, but proof
is lacking
 application of the principles of competition to natural
situations is not easy
 microbial situation in ecosystems is usually controlled by
heterotrophs competing for organic substrates as both
carbon and energy sources
 if you know the factors affecting microbial composition
of the microbiota, you can manipulate it
(2) Competition for Chemicals
or Available Energy
 All microorganisms require iron for growth
 siderophore: low mw ferric ion-specific chelating
agents
 dissolve precipitated Fe and make it available for
microbial growth
 siderophores scavenge Fe and make it unavailable to
other species
 this occurs at tissue level
 probiotics producing siderophores can outcompete
pathogens for Fe, thus limiting pathogen growth
 works best with pathogens that also produce
siderophores (e.g., V. anguillarum)
 (3) Competition for
Adhesion Sites
 Competition for gut adhesion sites would limit
colonization
 adhesion to enteric mucus is necessary for bacteria
to become established in fish intestines
 this is probably the first probiotic effect
 adhesion can be specific (based on adhesin and
receptor molecules) or non-specific (based on
physiochemical factors)
 total probiotic effect is probably a mixture of site
competition, production of inhibitory compounds and
nutrient/energy competition
 (4) Enhancement of
Immune Response
 Rem definition of an immunostimulant? Chemical
compounds that activate the immune systems of
animals and render them more resistant to
infections by viruses, bacteria, fungi and parasites.
 Immune response varies in animals
 lactic acid bacteria administered orally may induce
increased resistance to enteric infections
 problem: only shown with specific cell compounds
or dead cells
 good indications, but no proof
(5) Improvement of Water
Quality
 Proposed as a mode of action as a result of
monitoring water quality after addition of
probiotics
 usually associated with Bacillus sp.
 Hook: gram + bacteria are better converters
of organic matter back to CO2 than gram -
 thus: phytoplankton blooms are more easily
maintained (interesting research area!)
 monitor: DOC, POC
 so far, hasn’t been demonstrated
Rationale for
Selecting
Probiotics

Verschuere et al. (2000)

 Background Info

 Profound knowledge of culture

 Critical review of available literature
 Characterization of abiotic and biotic
environment impacting culture
 Must know relationship between
microbiota and host well-understood
 Relationship between microbiota and
carrying capacity of environment
Acquisition of Putative
Probiotics
 Are putative probiotics (isolated from host or
host environment) better than those from other
sources?
 Putatives might be better due to fact they are
already adhered to gut wall (i.e., could block
pathogen adhesion)
 If a non-pathogenic bacterium exists at high
density in typical culture water, it will be well-
adapted to prevailing conditions and can
probably compete efficiently with pathogens for
nutrients
 Screening of Putatives:
In-vitro antagonism testing
 Candidates are exposed to pathogen in liquid
or solid medium
 screened for production of inhibitory
compounds (bacteriocins), siderophores, or
on competition for nutrients
 must be undertaken with extreme caution:
not all media are suitable
 probionts are finicky as to on which medium
they produce inhibitory compounds (e.g.,
marine agar vs. TSB)
 Evaluation of Pathogenicity
 Probiotics must not be pathogenic to the host
organism -- this must be confirmed prior to
acceptance
 host must be challenged under stressed and non-
stressed conditions
 usually accomplished by adding probiotic to the
culture water
 proper way to do this under monoxenic conditions
(only the probiont present)
 also look at interaction with other food organisms
found simultaneously in culture (e.g., algae)
 In-vivo Evaluations
 Involves introducing candidate species to
host cultures and monitoring growth, survival,
physiochemical parameters
 means of addition: addition to artificial diet,
addition to culture water, bathing, addition
via live food
 next step: experimental (allochthonous)
infection of host via immersion
 needs long-term evaluation (is the pathogenic
effect one of suppression or delay?)
 Conclusions/Future
 Current status of probiotics in aquaculture is
really hazy
 Lack of knowledge on modes of actions is
very evident
 more info on competitive processes between
bacteria is required
 more info on relationship between bacteria
and other microbiota required
 economic value/efficiency, anyone???